Project Summary - Project 1 Cancer metastasis is a highly inefficient process. Past investigation supports the concept that formation of solitary tumors as metastatic deposits often takes months to years despite the fact that dormant cells are prevalent at the metastatic sites. Using genetically tagged human PC cells as models, we discovered that a small number of Metastasis-Initiating Cells (MICs) can recruit and reprogram genetically tagged non-metastatic PC cells (referred to as bystander or dormant cells) to participate in cancer bone and soft tissue metastases. MICs have the phenotype of expressing RANKL+, RANK+, c-MET+, p-c-MET+ (an activated form of c-MET) and are metastatic. Bystander cells being recruited by MICs are initially RANKL-, RANK+ c-MET-, p-c-MET-, and are non-metastatic, but after recruitment by MICs, formed solitary tumors at metastatic sites, acquired MICs phenotypes and became metastatic, even when injected in mice in the absence of MICs (Appendices 1 and 2). We found that cells with MIC and bystander cell phenotypes are in the blood circulation (CTCs or circulating tumor cells) freshly harvested from PC patients. Intriguingly, we have also shown that MICs can also reprogram CTCs and disseminated tumor cells (DTCs) isolated from PC patients. In support of the model studies, RANKL, p-c-MET, and NPR-1, a VEGF co-receptor and a downstream target of RANK-mediated signals, was confirmed in primary human PC tissues, and shown to predict the overall survival of PC patients. In this study, we will test the hypothesis that cells with MIC phenotype can recruit and reprogram bystander CTCs/DTCs to participate in PC metastatic cascade. We propose three Specific Aims to test this and related hypotheses: Specific Aim 1: To test the concept that circulating CTCs and DTCs serve as depots for MIC recruitment, initiation and reprogramming and contribute to cancer bone and soft tissue metastases. We will test the hypothesis that MICs coerce CTCs/DTCs to amplify a RANK-mediated signaling network through a feed-forward mechanism. Cells with an amplified RANK network may be able to colonize the bone. Specific Aim 2: To determine the molecular features of MICs and bystander cells required for successful recruitment, initiation and reprogramming to participate in cancer bone metastasis. We will test the hypothesis that freshly isolated and ex vivo expanded CTCs/DTCs must express RANK prior to be recruited, initiated and reprogrammed by MICs to express an activated RANK-mediated signal network. Specific Aim 3: To discover promising therapeutic targets at the interface of the tumor- microenvironment, and to test small molecule inhibitors and antibodies targeting relevant transcription factors (TFs) and their effectors in the signal pathways. We will test the hypothesis that TFs are coordinately activated in PC cells to develop the metastatic phenotype. Targeting these TFs (or their downstream effectors) could be highly effective in inhibiting the reprogramming of metastatic PC cells.